The invention concerns a method for nuclear magnetic resonance (NMR) spectroscopy of a sample comprising preparation of the sample and carrying out an NMR spectroscopy measurement. A method as described above is known from reference 8.
It has been shown recently1, 2 that it is possible to excite and sustain long-lived states (LLS) in systems comprising at least two coupled spins I=S=½. These LLS correspond to a difference between the populations of the anti-symmetric singlet state S0=N(|αβ−|βα) (where N=2−1/2) and the mean population of the three symmetric triplet states T+1=|αα, T0=N(|αβ+|βα), and T−1=|ββ. In order to sustain LLS, the spins must be rendered magnetically equivalent, either by removing the sample from the magnetic field, or by applying a suitable radio-frequency (rf) field. Typically, a continuous rf irradiation with an amplitude ω1=γB1, using an rf carrier placed half-way between the two chemical shifts, ωRF=(ω1−ωS)/2 can sustain LLS. These LLS correspond to diagonal elements of the matrix representation of the Liouvillian that describes the time-evolution in the presence of the rf field. Because the eigenvalues λLLS=RLLS=1/TLLS are real, the LLS relax mono-exponentially without oscillations. The lifetimes TLLS of LLS can be much longer than the spin-lattice relaxation time (TLLS>>T1), in favourable cases as much as a 36 times longer3. Spin polarisation has been preserved for tens of minutes using LLS in nitrous oxide4.
ELF Experiments
Levitt and co-workers discussed the behaviour of coherent superpositions of symmetric and antisymmetric states in a slightly different context than the present invention. In their ingenious experiments, the Zeeman polarized sample was taken out of the magnetic field, which in our language brings about a transformation from the product base into singlet-triplet base. In a vanishing magnetic field, it is possible to bring about a Rabi nutation of the two-level system comprising the S0 and T0 states by irradiation with an electromagnetic field at extremely low frequency (ELF)5, typically on the order of a few tens of Hz. In the case of 15N2O, they determined the scalar coupling J(15N, 15N) with unparalleled accuracy, since the linewidths are on the order of a few mHz, and the corresponding lifetimes on the order of 100 s.
‘Transverse Relaxation Optimized Spectroscopy’ (TROSY)
Wüthrich and co-workers made significant progress in improving the spectral resolution by seeking to extend the lifetimes of coherences. Their idea, which has become known under the acronym TROSY (for Transverse Relaxation Optimised Spectroscopy), was to use the mutual cancellation (negative interference) between the dipole-dipole (DD) and chemical shift anisotropy (CSA) interactions to trim relaxation rates6. In rapidly rotating side-chain methyl protons, there are similar beneficial interference effects between dipolar interactions7. The TROSY method has brought the threshold of molecular sizes up to which NMR is useful for structural biology of single-domain proteins from ca. 20 kDa to ca. 50 kDa. The use of selective deuteration techniques can help to further extend lifetimes of single-quantum coherences, since the number of dipolar interactions between the observed nuclei and other spins with high gyromagnetic ratios is reduced and, therefore, the remaining nuclei have slower relaxation rates. The combination of TROSY with selective deuteration has increased the molecular weight threshold considerably: recently, an NMR analysis of the dynamics in the 670 kDa 20S proteasome core particle was reported8.
Bio-NMR
The main strengths of nuclear magnetic resonance (NMR) lie in its atomic resolution and its non-invasive nature. To extract information from different sites the signals have to be resolved, i.e., have a frequency separation larger than their linewidth. As the linewidths are proportional to the decay rate constant of the observed spin coherence, the above requirement amounts to creating spin coherences with slow relaxation rates. This has been one of the main goals of liquid-state NMR spectroscopists for the last decade, ever since it was established that this would allow us to analyse more complicated molecules.
Object of the invention is to further improve spectral resolution in nuclear magnetic resonance sperocopy measurements.